The development of biologically derived therapeutics including proteins, cell receptor specific peptides, monoclonal antibodies [58], siRNAs and oligonucleotides designed to inhibit translation of key components of a signal transduction pathway [1, 59, 60], growth factors for improving cellular repair [61], and/or therapeutic cells to rebuild damaged tissues [8, 14, 62] is ongoing. While all biologically derived therapeutics are subject to delocalization after site-specific local injection or application, the macromolecules can be easily degraded by host proteases or ribonucleases. In addition, naked therapeutic cells undergo dramatic cell death such that only <3% cells are present shortly after transplantation [11, 20, 63]. One way to deliver these therapeutics to a specific locus is to mix them with hydrogels which not only protect these biologics and cells due to the polymer pore size, but also provide a lifelike cellular microenvironment rich in hyaluronic acid [21, 57, 64-66].
Hydrogels are three-dimensional hydrophilic, polymeric networks capable of imbibing large amounts of water or biological fluids. The networks may be comprised of homopolymers or copolymers [76]. While being highly hydrophilic, hydrogels are prevented from dissolving due to their chemically or physically crosslinked network. Water or biological fluids can penetrate between the polymer chains of the network causing swelling resulting in hydrogel formation. Hydrogels are appealing for biological applications because of their high water content and their biocompatibility [77]. Synthetic hydrogels provide a delivery vehicle for a wide variety of therapeutics including large molecular weight protein and peptide drugs as well as cellular based therapeutics.
Hydrogels from many synthetic polymers such as poly(hydroxyethyl methacrylate) (PHEMA), poly-(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) have been described [77]. Hydrogels created from naturally sourced material such as collagen, hyaluronic acid (HA), fibrin, alginate, agarose and chitosan have also been described [78].
HA is a glycosaminoglycan that is comprised of repeating disaccharide units and is prevalent, for example, during wound healing and in joints. Covalently crosslinked hydrogels formed by various chemical modifications have been described [79-84].
The preclinical use of hydrogels to maintain bioactivity and slow release of biologics has been described [15-19]. Furthermore, hydrogel use in cell delivery has been shown to improve cell viability and localization post-implantation [20-22]. Several different hydrogels have been used as excipients in FDA-approved ocular small molecule therapeutics to increase their residence time on the eye surface [23]. In addition, two new hydrogel formulations have been reported which show promise in delivering therapeutic cells to the subretinal space [6, 24-26]. While some of these formulations are composed of hyaluronic acid to match ocular tissues and maximize biocompatibility, these hydrogels do not have all of the characteristics required for successful delivery of both complex, fragile macromolecules, and cells.
Recently, a hydrogel based on thiol-modified derivatives of hyaluronic acid (HA) and porcine gelatin crosslinked with polyethylene glycol diacrylate (PEGDA) (trade name HyStem®) has been developed to meet these criteria [27, 30-33]. Crosslinked HA hydrogels, including HyStem®, have been successfully used in animal models of corneal epithelial wound healing [25], corneal tissue engineering [5], and retinal repair [7]. Crosslinked HA hydrogels also provide a flexible platform, allowing a user to modulate both gel compliance and gelation time by adjusting the ratio of its components [31, 34]. Since HyStem® gelation times are inversely proportional to final gel stiffness, higher concentrations of the PEGDA crosslinker will cause HyStem to gel in five minutes (G′>1300 Pa) while low concentrations require approximately one to two hours to form softer (G′<50 Pa) gels [31, 34, 35].
There are instances, however, when a modification of the HyStem® hydrogel composition is needed to retain both low compliance and rapid gelation time in a variety of applications. For example, corneal application would benefit from a softer hydrogel that gels within five minutes to prevent washout from the eye surface due to tear turn over and blinking [4, 23]. For example, therapeutic retinal progenitor cells require a low compliance gel to retain function [7]. A quick-gelling hydrogel would also aid in localizing the cells shortly after injection, preventing exudation through the needle track [36].
Thus there is a need for improved HA based hydrogels that can be readily tailored to meet specific applications, including therapeutic applications and the use of hydrogels for delivery of therapeutic agents. There is also a need for improved methods of making HA based hydrogels that provide for greater control of the physical and chemical characteristics of the hydrogel including, but not limited to, in situ gelation speed, cytocompatibility, biocompatibility and capacity to be functionalized. Moreover, there is also a need to simplify the manufacture of hydrogels in a cost effective way. The invention described herein meets these needs as well as other needs in the field.